PROJECT SUMMARY A pathogen?s ability to adapt to different environments is an important factor in its survival, infection, and persistence within its host. As a strict human pathogen, Streptococcus pyogenes (Group A Streptococcus, GAS) causes over 500,000 deaths annually. Even though it commonly manifests as superficial infections (e.g. strep throat), GAS also causes life-threatening diseases (e.g. necrotizing fasciitis, toxic shock syndrome) when it invades normally sterile tissues from initial sites of infection. It is imperative to gain a better understanding of GAS pathophysiology and the genetic requirements it needs to infect varying host environments. Our lab performed an in vivo transposon sequencing (Tn-seq) screen in a clinically relevant M1T1 5448 GAS strain to define the genetic requirements needed for GAS fitness in the subepithelial tissue, where we found a previously uncharacterized locus (scfCDE) to be essential for this environment. Based on homology alone, scfCDE are predicted to encode for a putative ABC importer. Because the locus has not been experimentally examined before, this proposal will explore the functional roles of scfCDE in GAS cellular pathways and virulence, hypothesizing that scfCDE encode membrane-associated proteins that play integral roles in GAS fitness in host tissue, importing a substrate into the cell important for GAS pathophysiology. This study will examine the scfCDE locus for phenotypes in nutrient utilization and in stress-induced environments to identify potential substrates these proteins may transport, explore its genetic architecture, expression, and regulation of the operon, and lastly, explore whether scfCDE are important for colonization, growth in human blood, and innate immune evasion using established in vivo and ex vivo models of GAS infection. Since scfCDE are conserved in GAS and other Firmicutes, successful completion of this project will potentially contribute to the development of novel therapeutic strategies against GAS and other important Gram+ pathogens.